When Steve Austria vacated his seat to run for the Ohio Senate, DeWine was elected to represent the 76th district in the Ohio House in 2000, defeating Democrat Eric Marcus and independent candidate Christina Turner, winning 62.3% of the vote.[3] In 2002, in the 70th district after redistricting, he defeated Democrat Lawrence Gordon
with 71.7% of the vote.[4] He faced Gordon again in 2004, DeWine won with 69.3% of the vote.[5]

In 2006, DeWine won his fourth term with 60.4% of the vote against Democrat Kevin O'Brien.[6] DeWine was elected Speaker pro tempore in the 127th Ohio General Assembly. He was the chair of the Rules and Reference committee, and also serves on the Finance & Appropriations and the State Government & Elections Committees. He was not eligible to run for a fifth term due to term limits.

On January 16, 2009, he was elected unanimously as chairman of the Republican Party of Ohio.[7] He was reelected as chairman in 2011. He was replaced in April 2012 by Robert T. Bennett, following DeWine's resignation over disputes with Ohio Governor John Kasich.[8]